Diabetic nephropathy (DN) is the most common cause of end-stage renal disease (ESRD) in the world and affects about 15-25% of type I diabetes patients and 30-40% of patients with type II diabetes. Clinical DN evolves in a sequence of stages initially with increases in glomerular filtration rate (GFR) and intraglomerular capillary pressure, glomerular hypertrophy and microalbuminuria. Poor glycemic control further exacerbates the disease progression to proteinuria, nodular glomerulosclerosis and tubulointerstitial injury and a decline in GFR which can eventually lead to ESRD. The development of DN is thought to result from the cumulative interactions among multiple metabolic and hemodynamic factors which activate intracellular signaling pathways that trigger the production of cytokines and growth factors leading to fibrotic renal disease. Although TGF- undoubtedly contributes to renal fibrosis, therapeutic attempts to neutralize it are potentially problematical due to the pleiotropic (some beneficial) effects of TGF- in normal tissues. Moreover, there are other pro-fibrotic agents implicated in DN that represent novel therapeutic targets. One such target is marinobufagenin (MBG), a cardiotonic steroid that has been implicated as a mediator of fibrosis in renal and cardiac diseases. The 3E9 monoclonal antibody (mAb) produced by a murine hybridoma neutralizes MBG and has anti-fibrotic activity in various in vitro assays and in rodent models of renal and cardiac fibrosis. The overall goal of this projec is to develop a humanized 3E9 mAb (hu3E9) as a therapy for diabetic nephropathy. The murine 3E9 has been cloned, expressed in mammalian cells and its biological activity confirmed. The first aim is to humanize 3E9 and if necessary use affinity-maturation technology to increase its binding affinity and specificity for MBG. A CHO cell line stably expressing hu3E9 will be developed. Finally, 3E9 will be evaluated in a mouse model of DN induced by streptozotocin treatment of mutant mice deficient in endothelial nitric oxide synthase. The results of this study will establish the feasibility of future preclinical and clinical development of hu3E9 as a novel therapy for DN and possibly other fibrotic diseases.